Nephroprotective effect of magnesium isoglycyrrhizinate against arsenic trioxide-induced acute kidney damage in mice

Abstract

Magnesium isoglycyrrhizinate (MgIG) has anti-inflammatory, antioxidative, antiviral and anti-hepatotoxic effects. However, protective effects of MgIG against renal damage caused by arsenic trioxide (ATO) have not been reported. The present study aimed to clarify the protective function of MgIG on kidney damaged induced by ATO. Other than the control group and the group treated with MgIG alone, mice were injected intraperitoneally with ATO (5 mg/kg/day) for 7 days to establish a mouse model of kidney damage. On the 8th day, blood and kidney tissue were collected and the inflammatory factors and antioxidants levels in the kidney tissue and serum were measured. The expression of protein levels of caspase-3, Bcl-2, Bax, Toll-like receptor-4 (TLR4) and nuclear factor-κB (NF-κB) were determined via western blot analysis. In the renal tissue of mice, ATO exposure dramatically elevated markers of oxidative stress, apoptosis and inflammation. However, MgIG could also restore the activities of urea nitrogen and creatinine to normal levels, decrease the malondialdehyde level and reactive oxygen species formation and increase superoxide dismutase, catalase and glutathione activities. MgIG also ameliorated the morphological abnormalities generated by ATO, reduced inflammation and apoptosis and inhibited the TLR4/NF-κB signaling pathway. In conclusion, MgIG may mitigate ATO-induced kidney damage by decreasing apoptosis, oxidative stress and inflammation and its mechanism may be connected to the inhibition of TLR4/NF-κB signaling.

Keywords: TLR4/NF-κB signaling pathway; arsenic trioxide; magnesium isoglycyrrhizinate; oxidative stress.

Figure 1

Chemical construction of magnesium isoglycyrrhizinate.

Figure 2

Effects of MgIG on levels of serum (A) BUN and (B) CRE and urine (C) CRE and (D) creatinine clearance. Values are demonstrated as mean ± SEM, n=10. ^**P<0.01 vs. CONT group; ^##P<0.01, ^#P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; BUN, blood urea nitrogen; CRE, creatinine; CONT, control group; ATO, arsenic trioxide group.

Figure 3

Effects of MgIG on histopathological variations in mice, as observed by hematoxylin and eosin staining [(A) magnification, x400; scale bar=50 µm]. The CONT group shows the standard structure of glomerular capillaries and tubular epithelium; the ATO group exhibits the inflammatory cell infiltration in the renal interstitial, glomeruli dilation and hyperemia; the MgIG treatment group shows a standard structure; and the H-MgIG and L-MgIG groups alleviate renal morphological alterations. (B) A boxplot was used to depict the kidney injury scores, in which the band inside the box represents the median, and the bottom and top of the box represent the lower and upper quartiles, respectively. The circle and star represent the largest and smallest data respectively. Values are demonstrated as median ± range, n=10. ^**P<0.01 vs. CONT group; ^##P<0.01, ^#P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; CONT, control group; ATO, arsenic trioxide group; H-MgIG, high MgIG group (50 mg/kg/day); L-MgIG, low MgIG group (25 mg/kg/day).

Figure 4

Effects of MgIG on the expression levels of oxidative stress markers (A) SOD, (B) CAT, (C) MDA and (D) GSH in serum. Values are demonstrated as mean ± SEM. n=10. ^**P<0.01 vs. CONT group; ^##P<0.01, ^#P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; GSH, glutathione; CONT, control group; ATO, arsenic trioxide group.

Figure 5

Effects of MgIG on the expression levels of oxidative stress markers (A) SOD, (B) CAT, (C) MDA and (D) GSH in renal tissues. Values are demonstrated as mean ± SEM. n=10. ^**P<0.01 vs. CONT group; ^##P<0.01, ^#P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; GSH, glutathione; CONT, control group; ATO, arsenic trioxide group.

Figure 6

Effects of MgIG on the ROS levels in mice [(A) x200 magnification, scale bar=100 µm]. (B) ROS-positive cell area percentages in each group are shown. Values are demonstrated as mean ± SEM. n=6 ^**P<0.01 vs. CONT group; ^##P<0.01, ^#P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; ROS, reactive oxygen species; CONT, control group; ATO, arsenic trioxide group.

Figure 7

Effects of MgIG on the expression levels in renal tissues of IL-6 (A), IL-1β (B) and TNF-α (C). Values are demonstrated as mean ± SEM. n=10. ^**P<0.01 vs. CONT group; ^##P<0.01 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; IL, interleukin; TNF, tumor necrosis factor; CONT, control group; ATO, arsenic trioxide group.

Figure 8

Effects of MgIG on the expression levels of apoptosis indexes were determined by western blot analysis. (A) For each group, the fluorescence intensity of (B) caspase-3, (C) Bcl-2 and (D) Bax was analyzed by normalizing to β-actin. Values are demonstrated as mean ± SEM. n=3. ^**P<0.01 vs. CONT group; ^##P<0.01, ^#P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2 associated X; CONT, control group; ATO, arsenic trioxide group.

Figure 9

Effects of MgIG on the expression levels of TLR4, NF-κB (p65) and p-NF-κB (p-p65). (A and D) For each group, the intensity of (B) TLR4, (C) NF-κB (p65), (E) p-NF-κB (p-p65) and (F) p-p65/p65 were analyzed by normalizing to β-actin. Values are demonstrated as mean ± SEM. n=3. ^**P<0.01 vs. CONT group; ^##P<0.01, ^#P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; TLR4, Toll-like receptor-4; NF-κB, nuclear factor-κB; p-, phosphorylated; CONT, control group; ATO, arsenic trioxide group.

Figure 10

Mechanism of MgIG on ATO-treated nephrotoxicity. MgIG, magnesium isoglycyrrhizinate; ATO, arsenic trioxide; TLR4, Toll-like receptor-4; BUN, blood urea nitrogen; CRE, creatinine; NF-κB, nuclear factor-κB; ROS, reactive oxygen species; IL, interleukin; TNF, tumor necrosis factor; CAT, catalase; GSH, glutathione; SOD, superoxide dismutase; MDA, malondialdehyde; Bax, Bcl-2 associated X; Bcl-2, B-cell lymphoma 2.